The present invention relates to the field of thermal processing of biological matter to alter or kill the cells. More particularly, a rapid rise in temperature is employed in a manner which avoids denaturation of proteins while altering membrane properties.
It is well known that biological cells may be killed in a manner of Pasteurization, in which the time temperature product of a process is sufficient to denature cell proteins necessary for vitality. Other cell killing mechanisms are known which involve physical process, such as shear forces, ultrasonic cavitation, alteration in membrane properties through the insertion of pores, and the like.
A number of methods are known for reducing bacterial activity in liquids. Traditionally, a so-called xe2x80x9cPasteurizationxe2x80x9d process is employed, which operates by the principles of thermal denaturation of proteins to inactivate bacteria. Thus, the liquid is raised to a particular temperature for a proscribed duration, to effect a statistical reduction in the number of, or even elimination of all viable bacteria. In an effort to reduce a duration of the process, high temperatures may be employed, which raise the temperature of the fluid to, e.g., 150xc2x0 C. for 2-4 seconds under pressure, followed by a flashing (rapid boiling) to lower the temperature, thus limiting the duration of the treatment. Such systems thus require a very high temperature, and may alter a taste of a potable liquid or food product, such as is the case with milk. Depending on how the heat is applied, precipitation of proteins in the product or other physical changes may occur. In addition, the presence of oxygen during treatment may cause accelerated oxidation.
The heat treatment processes for fluid food products (e.g., milk) are applied for destroying disease-causing microorganisms, as well as inactivating microorganisms which may spoil the food. In many known processes, the bacterial reduction is a preservation technique which extends the shelf life, but sterilization is not achieved. Some of these pasteurization techniques involving heat treatment of food products, for instance, milk, are disclosed in USSR Pat. No. N 463,250 M KI A 23c 3/02 and N 427532 M KI 28 9/00 A 23c 3/02.
The most widely used Pasteurized technique involves subjecting food products to heat treatment as high as 65-75xc2x0 C. and exposing same to this temperature for a period of time of 30 minutes. This is the so-called long-term heat treatment. The second technique involves subjecting food products to heat treatment at a temperature of 70-75xc2x0 C. and exposing same to this temperature for a period of time of 2-4 minutes. The third technique involves subject food products to short term heat treatment at a temperature of 95xc2x0 C. and exposing same to this temperature for 30 seconds. The fourth technique includes ultra high temperature heat treatment. It involves subjecting food products to a temperature of 110-140xc2x0 C. and exposing same to this temperature for a period of time of 2-3 seconds. These treatment are thus based on a thermostability time-temperature relationship of microorganisms. Thermostable life-time is defined as a life-time of microorganisms at a given temperature. The higher the temperature, the shorter the thermostable period. An effective Pasteurization treatment thus subjects food products to heat treatment at a certain temperature for a period of time which is longer than the thermostable period.
These prior art techniques are generally directed toward the thermal denaturation of essential cell elements, they effectively cook the treated medium, including any biological organisms therewithin. Thus, proteins lose their tertiary structure, cells are killed, and heat labile components are adversely affected. Sediments may also be formed, which may necessitate regular cleaning of the system, especially any higher temperature portions, such as heat exchange surfaces.
Some of these drawbacks can be avoided by using the direct heat treatment, which heats the product by way of direct contact of the product subjected to Pasteurization with the heating medium, for instance, steam, rather than through a heat transferring surface of heat exchange equipment. This method eliminates release of the milk xe2x80x9cstonexe2x80x9d in the heating zone and lessens its appearance on other surfaces of the equipment. These known methods transfer the product into the Pasteurizer, and inject steam made from potable water to a desired temperature, for a desired period. The product is cooled and excess water from condensed steam eliminated. This technique allows a relatively quick heat treatment of the product, and has been found of particular use in ultra high temperature heat treatments. The technique avoids exposure to temperatures higher than a desired final temperature, and thus may limit sedimentation, which may appear, for example, as milk xe2x80x9cstonexe2x80x9d in a Pasteurization process. Where direct steam contact is used, it dilutes the medium, for example up to 30% of the product mass, with an ultrahigh temperature Pasteurization technique, which subsequently is often removed.
These known methods of Pasteurization strive to maintain laminar flow of milk during the process, and thus do not atomize the milk. As a result, these systems fail to raise the temperature of the bulk of the milk at a rapid rate, and rather gradually raise the bulk temperature to the Pasteurization temperature, at which the milk is maintained for the desired period. Of course, a small surface layer may experience rapid temperature rises.
Zhang, et al., xe2x80x9cEngineering Aspects of Pulsed Electric Field Pasteurizationxe2x80x9d, Elsevier Publishing Co. (1994) 0260-8774(94)00030-1, pp. 261-281, incorporated herein by reference, relates to Pulsed Electric Field Pasteurization, a non-thermal Pasteurization method. This method (as well as other biological treatment methods) may be combined with other methods, to enhance efficacy of the composite process, while avoiding the limitations of an excess exposure to any one process.
RU 2,052,967 (C1) relates to a rapid temperature rise bactericidal treatment method, similar to the present method, but intended to non-selectively kill organisms. Abrams et al, U.S. Pat. No. 3,041,958 relates to a steam processing temperature control apparatus. Wakeman, U.S. Pat. No. 3,156,176 relates to a steam Pasteurization system. Stewart, U.S. Pat. No. 3,182,975 relates to a steam injection heater, which employs impeller blades to mix steam and milk for rapid heating. Engel, U.S. Pat. No. 3,450,022 relates to a steam infuser for high temperature steam treatment of liquids. Nelson, U.S. Pat. No. 3,451,327 relates to a steam injector for a milk sterlizer. This device is intended to bring the milk to a high temperature, and thus allows thermal communication between the steam and milk prior to venting. De Stoutz, U.S. Pat. No. 3,934,042 relates to a system for treating beverages, including milk, beer, wine and fruit juices, for sterilization or Pasteurization. The liquid is held at elevated temperatures for extended periods. Janivtchik, U.S. Pat. No. 4,160,002 relates to steam injectors for Pasteurizing milk using pressurized steam. Wakeman, U.S. Pat. No. 4,161,909 relates to an ultrahigh temperature heating system for heating, e.g., milk. The milk falls in a curtain configuration in a steam chamber. The milk held at a high temperature after heating. Nahra et al. U.S. Pat. No. 4,591,463, and Nahra et al. Re. 32,695, incorporated herein by reference, relate to a milk ultra Pasteurization apparatus in which sheets of milk fall within a steam filled chamber for ultra high temperature Pasteurization. Bronnert, U.S. Pat. Nos. 4,787,304 and 4,776,268 relate to an infusion heating apparatus for sterilizing liquid food products, having a porous steam dispensing cylinder or diffuser located along a central axis of a treatment vessel. Sanchez Rodriguez, U.S. Pat. No. 5,209,157 relates to a diary preparation system which involves an ultrahigh temperature treatment step.
It is also well known to fuse cell membranes through the use of so-called fusion proteins, chemical agents, photonic effects, and possibly by application of heat. Cell fusion has been used to form hybrid cells or hybridomas, to insert cell surface proteins or to alter cell cytoplasmic chemistry.
The Rapid Thermal Cycle Processing (RTCP) Technology is relatively unexplored as a mechanism for treating of cells. It is known, however, that RTCP is capable of killing bacteria at temperatures below those which tend to denature bacteria.
The RTCP process, also known as MilliSecond Pasteurization (xe2x80x9cMSPxe2x80x9d) involves the heating of fluid droplets with saturated steam, at a high rate of increase, for example, over a thousand degrees per second, to a desired temperature, typically under conditions which do not denature (a chemical process which alters structure) proteins. When appropriately processed, fluids may be sterilized, without otherwise changing macromolecular structures.
RTCP technology has been proposed for the xe2x80x9cPasteurizationxe2x80x9d of milk, to kill bacteria and spores in the milk.
A microwave Pasteurization and Sterilization process is disclosed in Stanley E. Charm et al. (Charm Sciences, Inc., Malden Mass.), U.S. Pat. Nos. 4,839,142, 4,975,246, and 5,389,335, expressly incorporated herein by reference. These patents disclose a process which is said to sterilize food products without substantial protein denaturation by rapid heating (25-8000xc2x0 C. per second) and cooling of the treated product within a short time.
The present invention provides a rapid thermal cycle processing system which provides a high rate of temperature change, which primarily is directed to formed cell components, such as membranes. In particular, the outer cell membrane is a focus of the action.
While the mechanism of action is subject to speculation, it is believed that a primary effect of the rapid temperature transition is to generate a shock wave. In addition, the rapid rate of temperature rise is fast enough that diffusion of lipids in the membrane is incomplete, so that domains of membrane remain with differing characteristics, such as glass transition temperature. Thus, the temperature rise may have a different qualitative effect on certain regions as compared to others.
While RTCP is capable of killing cells, likely by disrupting membranes, the RTCP process, need not always be applied under such conditions as kill the cells. This readily apparent from studies which were performed in which a surviving fraction of bacteria remained. In some tests, these bacteria formed aberrant colonies when cultured. Thus, by subjecting the cells to RTCP under mild conditions, alterations may occur which are non-lethal.
This observation has presented many significant and exciting opportunities. These opportunities include cell membrane fusion, presentation of cellular antigens for immune response, induction of specific cell responses, and cellular xe2x80x9creprogrammingxe2x80x9d. RTCP also has potential industrial applications in the biotechnology industry for sterilization, cell lysis, cell manipulation and cell fusion, and in the chemical industry for rapid and uniform heating, selective melting, reaction initiation and encapsulation or trapping of particulates.
The present invention seeks to alter cell characteristics by a thermal shock process, which may be used, for example, to inactivate or kill bacteria, alter cell surface chemistry or antigenicity, disrupt membranes, activate cell functions or responses, disaggregate cells, as a pretreatment before cell fusion or infection, activate or change the function of a cellular parasite (bacteria, mycoplasma, virus, prion, etc.), affect mitochondrial functioning or the functioning of other organelles. On an organism level, the present invention may be used to treat bacterial infections, such as osteomyelitis, vital infections such as AIDS, human or animal Herpes viruses (including HHV-5 and EBV, as well as CMV, HSV-1, HSV-2, VZV, HHV-8, and the like), treat cancer, sarcoma, mesothelioma, teratoma or other malignancy or neoplasm, treat skin conditions, such as psoriasis, treat inflammation, treat fungal diseases, blood borne diseases, leukemias and the like. The present invention may also have utility in the treatment of syndromes, which may be multifactorial in origin and involve an immunological component or defect. Therefore, the present invention may also find utility in the treatment of chronic fatigue syndrome (CFS), for example by applying immune stimulation therapy through treatment of blood or blood components.
The broad utility of the present invention comes from its ability to carefully control a stress applied to a cell. This stress may, of course, kill the cell or selectively kill a subpopulation of cells, but more importantly, it is believed that the present invention may be applied to cells to have a measurable non-transient effect which does not immediately result in cell death. In this manner, the present method provides a new manipulation modality for cells.
In contrast to known cellular thermal inactivation methods, the major aspects of the present invention do-not rely on thermal denaturation of cellular proteins and enzymes, but rather on a rapid temperature rise which irreversibly changes the cell, at temperatures and energy levels below those required by traditional Pasteurization processes.
In particular, according to one embodiment of the system and method according to the present invention, a product is treated such that the temperature of a medium in which all or a portion of the cells exist rises at such a rapid rate that normal accommodation mechanisms, which might allow the cell to avoid permanent effect from a slower temperature rise rate treatment, are unavailable or ineffective. Thus, it is an aspect of the present invention to alter cell functioning based on a rate of temperature change during treatment, rather than based on a time-temperature product function or a maximum temperature.
The present invention is thus believed to operate by a physical principle different than thermal denaturation, the principle behind Pasteurization. Rather than a thermal denaturation of the proteins, as well as proteins which may be in the extracellular medium, one aspect of the present invention operates by thermal shock, which is believed to disrupt or alter membrane structures or membrane components of cells. Typical media for treatment include milk, egg white, blood plasma, cell culture medium, fermentation broth, fruit juices, and the like.
Thus, rather than a high temperature, per se, the present invention requires a high rate of temperature rise. The resulting maximum temperature may be limited to temperatures which do not denature various proteins, e.g., a maximum temperature of 0-75xc2x0 C. It is clear, therefore, that the maximum temperature may remain sub-physiological, or rise to relatively high levels. For food processing, the maximum temperatures will often be on the higher end of the scale, in the 40-75xc2x0 C. range, while in medical or pharmaceutical process, the maximum temperatures will often be in the middle of the range, e.g., 15-55xc2x0 C. Sterilization of non-heat labile media may occur at high temperatures, e.g., greater than 110xc2x0 C., for example where the media is contaminated by thermotrophic organisms.
One theory of operation of the present invention relates to the glass transition temperature of membrane structures. Cellular membranes are generally formed of phospholipid bilayers with proteins, lipoproteins and glycoproteins inserted on the inside, outside, or protruding through the membrane. The membrane, especially the fatty acid chains of the phospholipids, are physiologically maintained in a fluid condition, and thus lipids and proteins are motile across the surface of the membrane. For example, under comparable circumstances, a lipid molecule may travel at a rate of about 2 microns per second, with proteins traveling at a rate of several microns per minute, in the plane of the membrane. Membrane components, though mobile in the plane of the membrane, are generally slow to switch or invert between the outer and inner surface. For example, transverse diffusion rate of phospholipids is about 10xe2x88x929 the rate of lateral diffusion, for a typical 50 xc3x85 distance (the thickness of a phospholipid bilayer membrane). The viscosity of a cell membrane typically is about 100 times that of water.
On the other hand, the membrane structures of living cells have some long-term ordering of molecules, especially the structures on the surface of the membrane (as opposed to the lipid phase in the middle of the membrane), and therefore are in this sense somewhat crystalline. Thus, the phrase xe2x80x9cliquid crystalxe2x80x9d is apt for the composite structure. Among other functions, the controlled membrane fluidity is believed to be necessary for various mediated transport systems which involve the movement of carriers within or through the membrane. The membrane proteins also have, in their natural state, a separation of charged and uncharged portions, allowing stable insertion of lipophilic portions of the proteins into the membrane structure, with hydrophilic portions protruding extracellularly or intracellularly from the membrane, into the cytoplasm or extracellular fluid. Intracellular membranes may also have asymmetry. Since the phospholipids are essentially undistinguished, the long term (i.e., over distances of tens of Angstroms) ordering of the membrane along its surface is related to arrangements of the protein components and the polar end-groups of the phospholipids. Some of the proteins or protein structures which extend through the membrane provide channels which allow ions, such as sodium, potassium and chloride to readily cross, or to be selectively controlled or pumped. The size of the channel allows selectivity between differing ions, e.g., sodium and potassium.
The tertiary configuration of the proteins (the three dimensional structure of a single protein molecule), and quaternary configuration of peptide structures (the spatial interaction of separate molecules) are thus critical for proper protein insertion in the membrane, and protein functioning. Thus, the membrane is ordered, and this ordering relates to its function. A disruption of the ordering affects the cell function, and may destroy the cell, or have a lesser damaging, distinct or selective effect.
The membrane fluidity may be controlled by fatty acid composition. For example, bacteria use this mechanism. The fatty acid chains of lipid molecules may exist in an ordered, crystal-like state or in a relatively disordered fluid state. The transition from the ordered to disordered state occurs when the temperature is raised above a xe2x80x9cmeltingxe2x80x9d temperature, or more properly, a glass transition temperature. In the case of fatty acyl chains within the membrane, the physiological state is fluidic. Of course, the membrane structure may have a number of different glass transition temperatures, for the various components and their respective energetically favorable orderings which may exist. This glass transition temperature depends on a number of factors, including the length of the fatty acyl side chain and their degree of unsaturation. Unsaturation (with the naturally occurring cis-oriented carbonxe2x80x94carbon bonds) xe2x80x9c causes kinksxe2x80x9d in the side chains, and increases bond rotation on either side of the unsaturation, both of which impair orderly packing, thus reducing crystallinity and increasing the glass transition temperature. Long fatty acyl chains interact more strongly, stabilizing the structure, and in increase in their proportion leads to a decrease in glass transition temperature.
Higher organisms have cholesterol in their membranes, which increases membrane fluidity. The cholesterol content may be controlled to control fluidity.
It is known that in E. coli, the ratio of saturated to unsaturated fatty acyl chains in the cell membrane decreases from 1.6 to 1.0 as the temperature decreases from 42xc2x0 C. to 27xc2x0 C. This decrease in the proportion of saturated residues is believed to prevent the membrane from becoming too rigid at lower temperatures. Higher species, including mammals, regulate cell membrane fluidity through cholesterol content, although this mechanism is believed to be absent in bacteria. It is believed that these membrane-composition accommodation mechanisms are comparatively slow.
It is also believed that organisms, such as bacteria, maintain their cell membranes a number of degrees below an important glass transition temperature of the membrane, thus assuring a balance between membrane fluidity and crystalline-like ordering. This crystalline state also implies a non-linear response of the membrane to temperature variations around the glass transition temperature.
Cellular mechanisms are believed to be present which assure that, through commonly encountered temperature variations, irreversible cellular damage does not occur. Some of these mechanisms are active or controlled, and thus have a latency. Some of these temperature changes may also trigger physiological cellular responses, such as so-called temperature shock proteins. Some of these mechanisms are physical and passive, and thus occur relatively rapidly. These include stretching, membrane shape changes, and the like.
According to this theory, the system and method according to the present invention seek to take advantage of these delayed responses in the accommodation mechanisms to temperature increases, by increasing the temperature, through this glass transition temperature, at such a rate that the cellular mechanisms do not have a chance to effectively respond, thus allowing irreversible damage to the bacteria, presumably through a disruption of the higher levels of organization, without necessarily affecting the lower organizational levels of structure. Thus, the temperature of the bacteria need not be raised to a temperature sufficient to thermally denature the tertiary structure of proteins.
Another theory for the observed bactericidal effect, and indeed the sterilizing effect believed to exist, is that, though the temperature of the cells is raised, it is not raised sufficiently to completely fluidize the membranes, leaving them comparatively stiff, brittle or non-compliant. The thermal shock according to the present invention also produces a mechanical stress, which may damage or affect the membrane. This damage may result in lysis, or a less severe mechanical disruption, which may later result in cell death or other response. This mechanical stress may also activate cellular processes or otherwise influence cell functioning. This effect is essentially opposite to that seen in high temperature Pasteurization (HTP), wherein the sustained higher temperatures tend to liquefy the membrane; although these HTP processes are specifically intended to thermally denature proteins to inactivate cells.
High temperature change rates are needed in order to prevent the relaxation of structural changes in a cell, e.g., the cellular membrane, which occur over approximately 10-100 mS. With temperature rise rates in excess of this rate, an effect occurs, which may, for example, disrupt or inactivate bacteria or cells or have other effects.
The induced thermal shock thus produces a number of effects on the cell. First, the cell rapidly expands due to the increase in temperature. Second, the cellular membranes may experience a configurational change either as a primary effect or secondarily due to a phase, volume or shape change of cellular components. Third, while thermal denaturation generally is directed to essentially irreversible changes in the tertiary protein structures of critical proteins and enzymes, thermal shock may effectively reduce quaternary organization to control or alter the cell. Microtubule structures and nucleic acid conformations may also be affected.
According to the present invention, one method for inducing this controlled vet rapid temperature rise is by treating medium containing the cells, generally in relatively small droplets to provide a large surface area to volume ratio and small thermal inertia held at a starting temperature, with an excess of steam at the desired final temperature. The interaction between the droplets and steam is rapid, equilibrating within milliseconds at the final temperature, with only a small amount of dilution due to the high latent heat of vaporization of steam. Generally, in order to reduce a rate-limiting boundary layer, the droplets are degassed prior to treatment.
The water derived from condensed steam chemically dilutes the droplets, rather than mechanically diluting them. In the case of milk, this means that the water is associated with the milk proteins, and the treatment does not substantially adversely affect the flavor of the milk. This excess water may also be removed. In the case of biological media, the dilution is relatively small, depending on temperature rise, and therefore is unlikely to induce a substantial hypotonic shock. However, to the extent that this hypotonic shock does induce a response, that response forms a part of the present invention.
Alternately, other controlled addition of energy to the cell-containing medium or tissue may be used. Thus, a microwave device may be used, which heats the medium through molecular excitation. The power of the microwave is controlled so that the medium is heated to a desired temperature over a desired period. The energy is applied rapidly, in order to obtain the desired temperature rise rate, e.g., in excess of 1000xc2x0 C. per second, over a short period.
The treatment may also be applied to tissues, since atomization is unnecessary. The use of rapidly applied microwave radiation also means that thermal diffusion or blood perfusion become comparatively insignificant factors in the treatment. The volume to be treated may be physically measured, estimated, or empirically determined by a xe2x80x9ctestxe2x80x9d treatment which applies a relatively small amount of energy and determines the temperature rise in response. In any case, it is important to assure uniformity of treatment of bulk tissues, in order to prevent spatial variations in treatment. However, where the goal is not treatment of all cells within the organ or tissue, for example and organ such as lung, liver or brain, or a tissue such as a solid tumor, then the treatment may be directed toward a portion of the tissue, with care taken not to over-treat any essential tissues. Thus, non-uniform or non-uniform fields of microwaves or infrared radiation (coherent or incoherent, monochromatic or broadband) may be employed to heat cells or tissues.
In general, visible or ionizing radiation and acoustic waves are not generally preferred energy sources because, in order to raise the temperature by the desired amount, other effects will likely be produced in the tissues. However, where these other effects are desired or complementary, they may be employed. For example, ionizing radiation may have a different mechanism for killing or altering cells, where this effect is desired.
A composite treatment may also be fashioned, in which a core tissue is destroyed, while a peripheral shell is partially treated. It is known that one mechanism by which neoplastic cells escape normal immunological surveillance is by hiding antigenic factors from the cell surface, or even not producing certain antigenic markers. It is believed that this aspect of the present invention will overcome these mechanisms are disrupt or alter membranes so that antigenic markers or elements are accessible. In this case, cell death is not necessary for efficacy, as the mere presentation of unique or characteristic antigens may be sufficient to spur an immunologic response which results in an effective treatment.
Blood presents certain interesting properties material to its application for treatment according to the present invention. First, it may be transferred to an extracorporeal reactor. Second, blood components may be separated in real time, in a plasmapheresis process, and individual blood components (erythrocytes, leukocytes, platelets, plasma, etc.) treated separately. Third, it is a liquid which maybe separated into small droplets. Thus, blood treatment may be effected through the steam chamber, using treatment parameters which do not coagulate or denature blood proteins. Generally, a useful blood treatment does not attempt to kill all blood cells, or one would simply extravasate without reinfusion, or separate undesired cell components and not reinfuse undesired components.
Therefore, often a goal of therapy is either selective treatment of a subpopulation of the cells, or a non-lethal treatment applied non-selectively to all or some of the cells present In order to effect a non-lethal treatment, the temperature rise rate is controlled, and/or the temperature rise and/or maximum temperature is controlled.
Blood treatments may be effective, for example, to treat chronic fatigue syndrome (CFS), acquired immune deficiency syndrome (AIDS), malaria, babesiosis, other viral, bacterial, fungal or parasitic diseases, leukemias or other blood-borne neoplasms, blood dysplasias and dyscrasias, immune disorders and syndromes. Bacterial-associated autoimmune mediated disorders, such as those related to spirochetes (syphilis, Lyme disease), as well as other autoimmune related diseases, such as rheumatoid arthritis and lupus, may also be subject to treatment according to the present invention. This later treatment may be applied, for example, to lymphocytes, which mediate immune responses.
In some syndromes, viruses play a primary or ancillary role. Many types of viruses have a lipid coat, which is, for example, derived from the cell membrane of a host cell before budding or lysis, generally with viral-specific proteins or glycoproteins. It is characteristic of chronic viral infections that the viruses avoid vigorous immunological response by not presenting antigenic proteins, or by mimicking host proteins. On the other hand, some chronic viral infections produce an autoimmune response which does not particularly target or eliminate viral infected cells. In either case, the temperature shock method according to the present invention allows relatively mild reconfiguration of membranes, allowing normally unavailable antigenic markers of membrane proteins or intracellular proteins to be presented to the host immune system. For example, a cell membrane inversion may take place, presenting internal antigens. Thus, any disease which is characterized by deficient or misdirected host immunological response is a candidate for treatment according to the present invention. Accordingly, cells which are accessible through the blood, skin or in particular organs may be targeted with the temperature shock treatment.
It is also noted that temperature shock may be used to redirect the activities of a cell. For example, circulating immune cells may be refractory or hyperstimulated. A treatment according to the present invention may be used to xe2x80x9cresynchronizexe2x80x9d or reset cells to obtain a normal response. Thus, the present invention need not be directed to the treatment or destruction of abnormal cells, but rather to the use of temperature shock for a variety of purposes.
Since the treatment of an individual patient does not necessarily require high throughput, other energy sources may be used, besides steam and microwaves, including general infrared, laser, maser, and chemical sources. Therefore, for example, a stream of blood, or blood component(s), may be subjected to a controlled low power CO2 laser or microwave treatment to effect the temperature shock treatment. Further, using cell separation techniques, such as those developed by Coulter Electronics, Hialeah, Fla., individual blood cells may be separated and individually treated, based on an identification of type, and then, for example, reinfused into the host.
The cell treatment methods according to the present invention may also be applied to in vitro techniques in order to control cells or select cell subpopulations. Typical applications include, for example, genetic engineering clonal selection for temperature shock resistance genes, which may be either a primary goal or a marker gene for a linked trait.
Another organ of interest is the skin, which may have tumors (malignant melanoma, basal cell carcinoma, etc.), psoriasis, viral, bacterial or fungal infection, inflammation, other immunological or autoimmune disorder, loss of elasticity, angioma, and other conditions. The skin is of particular interest because of the ease of external access to the surface. Therefore, for example, a stream of steam, laser beam or infrared source may be applied to the skin, in a manner which would quickly raise the temperature or the surface and possibly a region below the surface. In contrast to types of known treatments, the temperature rise is carefully controlled to avoid ablation or burning, while the heating is nearly instantaneous. The careful control is exerted, for example in the case of steam, by controlling the partial pressure of the steam and performing the treatment within a controlled environment, such as a hypobaric chamber or enclosure. In the case of laser, the pulse energy and repetition rate, as well as particular wavelength of the laser, e.g., CO2 with 10.6 xcexcm wavelength, may be empirically determined for an effective treatment. In the case of other electromagnetic waves, the field strength and duration of exposure are carefully controlled to effect a desired treatment.
The RTCP Process
The technologies include the use of an apparatus which atomizes a fluid to a uniform small droplet size and subjects the droplets to a treatment. The process treatment passes the droplets through a steam chamber at controlled temperature, which results in a rapid heating and thermal equilibrium of the droplets. The droplets are expelled from an atomizer nozzle at high velocity, so that the residence time in the steam chamber is limited. Since the maximum temperature is tightly controlled, this allows precise treatment parameters based on droplet size, velocity, steam temperature and pressure, and the presence of gasses other than water vapor. The treatment also is influenced by the deceleration of the droplets after treatment, a small dilution factor due to condensed steam, and the cooling of the fluid droplets after treatment.
The equilibration to the steam temperature is so rapid, in fact, due to the high latent heat of vaporization of steam, that a thermal shock is generated in the droplets. This rapid rise in temperature is accompanied by a mechanical expansion, which generates a mechanical shock wave in the droplet. The steam condensation also produces an osmotic xe2x80x9cshockxe2x80x9d, with up to 10-15% dilution, synchronized with the thermal-induced shock. Finally, the droplets are atomized, pass through the steam chamber at a velocity of about 20 meters per second, and are decelerated, for example by impact, resulting in a further synchronized mechanical shocks.
This technique has been shown to kill bacteria and spores with maximum process temperatures of 45-55C, under conditions which do not significantly denature milk proteins or most intracellular proteins. Thus, this technique is now being developed for commercialization as an effective milk sterilization technique. Bacterial cells subjected to this process and which survived were found to have persistent morphological changes in colony growth, which may indicate genetic or regulatory response to the RTCP process.
Using a Pasteurization system according to the present invention, many log reductions in E. coli were achieved, for example a reduction of from 106 per milliliter to the limit of detectability was achieved. Bacterial spores (B. subtilis) are also reduced, although possibly with lesser efficacy, for example a two log reduction (1% of original concentration) is achieved. It is believed that further refinement of the present system and method will prove more effective against these spores, and prove effective to kill or produce a response in various types of viruses, bacteria, fungi, protozoans, animal cells, and plant cells. The existence of organisms which survive treatment is clear evidence of the gentleness of treatment, and therefore that the treatment may be modulated to effect various survival fractions, and selective treatment of cell populations.
While strains which are desired to be treated may be found which are resistant to the system and method according to the present invention, supplemental methods may also be employed to treat the same medium, such as pulsed electric fields, oscillating magnetic fields, electron ionizing radiation, intense light pulses, actinic light or other visible or ionizing electromagnetic radiation, and high pressure treatments. Thus, treatments may be combined to effect complete Pasteurization or sterilization or more selective cell changes. See, Zhang et al., supra, Mertens et al., xe2x80x9cDevelopments in Nonthermal Processes for Food Preservationxe2x80x9d, Food Technology, 46(5):124-33 (1992), incorporated herein by reference.
The parameters of a bulk medium steam treatment process which control the efficiency include starting and ending temperatures, steam overheating, rate of temperature rise, degassing procedure (if any), pressure, pre- or post-treatments, pH, droplet size and distribution, droplet velocity, and equipment configuration. Presently, systems operable for milk Pasteurization have been tested using various parameters. For example a test has been conducted with a temperature rise from about 46xc2x0 C. to about 70.8xc2x0 C., with a milk pH of 6.60 (start) to 6.65 (finish), and a dilution of 2.5%. Droplet size is preferably about 0.2-0.3 mm. The rate of temperature rise is, for example, in excess of 1500xc2x0 C. per second, and more preferably above 2000xc2x0 C. per second. Under these conditions, with a starting bacterial and spore concentration of 10,000 spores per ml, the final concentration was 12 per ml. Thus, a reduction of about three logs was achieved under these conditions, without, for example, sedimentation of milk protein or noticeable alteration in taste.
The bulk medium steam treatment apparatus according to the present invention provides a rapid temperature rise by subjecting relatively small droplets of less than about 0.3 mm to dry steam (non-supercritical) at a partial pressure less than about 760 mm Hg. For example, with a low partial pressure of non-condensing gasses (e.g., less than about 100 mm Hg, and more preferably below about 50 mm Hg), the partial pressure of steam is about 0.3-0.8 atmospheres (e.g., about 225-620 mm Hg). The steam is saturated, and thus the temperature of the steam is held at a desired final temperature, e.g., 40-75xc2x0 C. The steam temperature-pressure relationships are well known, and need not be reviewed herein.
Droplets of medium including cells to be treated are atomized under force through a nozzle, into a reduced pressure reactor chamber containing the steam. Under this partial vacuum, residual gasses are drawn out of the droplet, which may form a boundary layer, reducing heat transfer rate; therefore, it is preferred that the bulk medium to be treated is degassed prior to treatment. Along its path, the droplets contact steam, which condenses on the relatively cooler droplets and heats the droplets through release of the latent heat of vaporization. As the steam condenses, the droplets are heated, until they reach the equilibrium temperature of the steam, at which tine there is no further net condensation of steam. The droplets will not get hotter than the steam in the chamber, so that the steam temperature sets the maximum temperature. However, depending on reactor configuration, the droplets may not reach equilibrium, and thus may reach a maximum temperature somewhat cooler then the steam. Of course, the initial interaction of the droplet with the steam will produce the highest temperature change rate, so that the reactor system may be designed to operate at a steady state which does not achieve equilibrium temperature. In this case, however, parameters should be tightly controlled to assure complete treatment without overtreatment, and thus a maximum temperature above a desired level.
The condensation of steam on the droplets induces pressure variations, or more properly steam partial pressure variations, within the reactor. In order to prevent a buildup of non-condensing gasses through outgassing or impurities, a vacuum pump may be provided which continuously withdraws gas, with a port near the droplet injection nozzle, removing the non-condensing gasses and some steam. Preferably, however, the product to be treated is fully degassed prior to entry into the reactor, and thus there will be little or no buildup of non-condensing gasses which require evacuation from the reaction vessel during processing. The droplet rapidly equilibrates with the steam temperature under the pressure conditions, over a distance of less than one meter, for example within 70 mm from the droplet injection nozzle. The so-treated droplets are then collected, and may be immediately cooled, thus limiting any adverse effects of long-term exposure to the steam temperature.
In one embodiment, the reaction vessel is provided with a number of zones which maintain steady state distinction. For example, in an initial portion, a low absolute pressure is maintained, degassing the droplets. In a subsequent portion, the droplets are contacted with steam, resulting in a rapid temperature rise of the droplets to effect the desired treatment. In a final section, a low steam partial pressure is maintained, allowing vaporization of water from the droplets, allowing flash cooling. In this manner, the time temperature product may be held at very low levels, effecting a rapid temperature increase followed by a relatively rapid temperature decrease. In order to provide separate temperature zones within the reactor, an external energy source within the reactor may be provided, such as infrared radiation source, to maintain steam temperature. Zones may also be separated by baffles which allow droplets to pass, while providing a gas flow restriction.
The steam in one preferred embodiment is provided by a steam generator, which boils, for example, potable or distilled water. This water is degassed prior to use, so that the steam contains few impurities and almost no non-condensing impurities. The steam generator may be at any temperature above the final temperature, e.g., 150xc2x0 C., as the thermal treatment of the droplets derives mainly from the latent heat of vaporization of the droplets, and very little from the absolute temperature of the steam. Preferably, the steam is saturated, which will define its temperature in a given atmosphere. If the steam is sub-saturation, condensation of steam on the droplets will be impeded. If the steam is supersaturated, it will itself form droplets and impede the process, in addition to diluting the medium. Process temperature control will also be adversely affected, and may be less predictable.
Thus, the mass flow rate of the saturated steam entering into the treatment system (in relation to the product flow rate and any withdrawal of steam or external heat transfer), controls the process treatment temperature. In the case of an over-pressure steam generator, the mass flow rate is restricted to prevent the treated droplets from reaching too high a temperature, or supersaturation conditions.
The steam is injected adjacent to the path of the droplets being treated, to ensure equilibration by the time the droplet reaches the terminus of the reactor. Due to boundary layer effects of the droplet, due to, for example, non-condensing gasses, as well as diffusion limitations, the temperature rise is not instantaneous. However, using the system in accordance with the present invention, it has been found that temperature rise rates in excess of 2000xc2x0 C. per second, or even 7600xc2x0 C. per second, may be achieved, which are sufficient to inactivate bacteria, and thus will effect may different types of cells and cellular structures.
It is noted that steam has a latent heat of vaporization of 540 cal/ml; therefore, a 5% ratio of steam to aqueous fluid to be processed will result in an approximately 27xc2x0 C. rise in temperature. The resultant 5% dilution may be inconsequential, or remedied in a later step.
In the bulk medium steam treatment device, the medium is sprayed through a nozzle as a stream of small droplets into a reaction vessel. The size of these droplets is preferably less than 0.3 mm, though if the droplets are too small they may present other difficulties, such as poor trajectory control, e.g., from low inertia loss of velocity, e.g., due to drag, Brownian motion, coalescence, and the like. Further, reduced droplet size may reduce potential throughput. In addition, since, if the droplet is too large, each drop of the medium may not be effectively treated, the droplet size distribution should include only a small umber of larger droplets, e.g. less than 1% of greater than 0.45 mm. Steam, which is produced in a steam generator, from, e.g., potable water, is supplied to the reactor vessel through a nozzle or array of nozzles. Steam condenses on the droplets, giving up its latent heat of vaporization to the droplets. The magnitude of heat transfer during condensation is very high, so that the speed of heating reaches several thousand degrees Centigrade per second. Therefore, in the several milliseconds it takes for droplets to travel through a reaction vessel, the temperature is raised substantially, effecting cellular alteration, e.g., bacterial inactivation, according to the present invention.
The steam is derived from a boiler. Tight control of temperature may require a high temperature boiler with a control valve near the reactor vessel. In other words, in order to ensure adequate flow of steam into the reactor, an excess capacity should be available from the boiler. Control is effected near the reactor, to avoid time response delays or oscillation. The water in the boiler is preferably degassed to eliminate non-condensable components. The boiler may have a superheater at its outlet, to heat the steam over a condensation equilibrium level.
The steam is injected into the reactor vessel through a number of steam injection ports, spaced along the path of the droplets within the chamber, so that the region distant from the fluid injection port maintains a relatively constant water vapor pressure. Thus, depending on the desired conditions, effective Pasteurization may be obtained with as low as between 2-5% by weight steam, condensed on the fluid droplets to achieve the temperature rise. There are temperature gradients in the reactor chamber, primarily near the injection nozzle. Since the steam condenses on the droplets, a partial vacuum is created in the region around the droplet, until the droplet reaches a temperature in equilibrium with the pressure, e.g., around 55xc2x0 C. at 0.5 atmospheres, and thus the vapor pressure equalizes. At equilibrium, the net condensation ceases, and the droplet remains in equilibrium.
The steam consumption is significantly lesser than in known ultra high temperature Pasteurization processes. Assuming the temperature of heat treatment applied to milk is to 60xc2x0 C., the temperature of milk upon entering the reaction vessel equal to 6-8xc2x0 C., then the required temperature rise will not exceed 55xc2x0 C. It will require about 55 Kcal for the heat treatment of one kilogram of fluid medium. As the condensation heat of one kilogram of steam is equal to 540 Kcal (540 cal/ml), only about 0.1 kilogram of steam will be required for condensation, i.e., only 10% of the mass of the product subjected to processing. Obviously, lesser temperature rises will require less steam.
The treated fluid medium, which has been slightly diluted with condensate, is collected on the bottom surface of the reaction vessel and then is supplied through a special vent into the discharging tank. The collected medium is subjected to a vacuum treatment, evacuating gases (air) and steam from the discharge, thus elating excessive water, cooling the fluid medium through water evaporation, as possibly through an external cooling system. The fluid medium may then, for example, constitute a final product or be used as part of a medical treatment.
In a typical bactericidal treatment system, a temperature rise of a liquid to be treated is from about 25xc2x0 C. to about 55xc2x0 C. For example, in a reactor 30 cm high, with a droplet velocity of 20 m/sec, the residence time will be about 15 mS. Thus, assuming an inlet temperature of 25xc2x0 C. and an outlet temperature of 55xc2x0 C., the temperature change is 30xc2x0 C. over 15 mS, or about 2000xc2x0 C. per second. Typically, the temperature rise will not be linear, nor will equilibration require the entire reactor length, so that the maximum temperature rise rate will be well in excess of 2000xc2x0 C. per second.
The liquid to be treated may be degassed prior to processing, to prevent accumulation of non-condensing gasses within the steam treatment reactor and resultant alteration of the thermodynamic operating point. Further, by degassing the liquid prior to interaction with the steam, the interaction with, and condensation of, steam on the fluid droplets is facilitated. Preferably, non-condensing gasses are kept to less than about 50 mm Hg, and more preferably less than about 20 mm Hg.
Alternately or additionally to degassing prior to dropletization, the fluid may be degassed within a first region of the reactor, under relatively high vacuum, after atomization, with subsequently reaction with the steam in a second portion of the reactor. Thus, due to the gas withdrawal in the upper portion, and condensation of the steam onto the relatively cooler droplet stream, steam will tend to flow from the second portion to the first portion of the reactor. Preferably, a baffle is provided between the two regions, with a relatively high density of fluid droplets to be treated, present in the transition region between the two portions, so that the steam condenses on the fluid droplets in this region, maintaining the pressure differential while effectively treating the droplets. Thus, steam vapor diffuses toward the fluid injection port.
In general since the treatment chamber vessel operates at a maximum process temperature below 100xc2x0 C., the pressure within the reactor will be below atmospheric pressure. For example, with an operating temperature of 55xc2x0 C., the chamber will be held at approximately 0.5 atmospheres, or 380 mm Hg. Thus, the pressure in the chamber determines the operating temperature: if the pressure is too high, the necessary temperature to achieve that vapor pressure of steam increases or steam condenses, raising the temperature of the reactor, and vice versa. This condition is considered xe2x80x9cwetxe2x80x9d. The control over processing is thus primarily exerted by the net mass flow of steam into the reactor. As stated above, a vacuum pump may be provided which exhausts non-condensing gasses and may also withdraw excess steam, allowing an additional control parameter and further allowing a non-equilibrium steady state to exist. For bactericidal treatment, non-condensing or xe2x80x9cdryxe2x80x9d treatment conditions are preferred.
The walls of the reactor vessel should be maintained at least at or slightly above the final operating temperature, to avoid condensation of steam on the wall and unnecessary product dilution. This may be done by any suitable heating system.
In fact, a number of methods are available to prevent droplets which are insufficiently treated due to, for example, coalescence into large droplets or statistical variations droplet size during atomization, from contaminating the treated product. For example, the droplets may be electrostatically charged, and then normally diverted from a direct path. Droplets of too large a mass will be diverted less, and may be separately collected. Alternately, an entire stream segment may be diverted if a flaw (untreated or untreatable portion) in the treatment is detected. For example, an optical detector may detect a large droplet traversing the reactor and divert the outlet for a period of time to flush any contaminants. An electrostatic or magnetic diversion system may also be employed to electrostatically charge droplets and then separate large droplets from small droplets.
It is believed that, as long as sufficient steam is present, small droplets will be effectively treated, while the persistence of bacteria contamination through treatment is believed to be related to the existence of large droplets. Thus, by eliminating or preventing large or untreated droplets, the effectiveness of the treatment is maintained.
The reactor may also include a second form of treatment, such as ultraviolet radiation, which may be supplied by ultraviolet lamps illuminating within the reactor. Since the droplets are small, the light penetration will be high, thus ensuring full coverage. Likewise, microwaves or other radiation may me used for auxiliary heating of droplets to the desired temperature, or for an electromagnetic field treatment of the droplets.
Where the fluid to be treated contains other volatile compounds, such as ethanol, such vapors may evaporate from the droplets, especially at elevated temperatures. This produces two effects. First, a boundary layer is created by the net outward mass flow, which may impede steam contact and heating. Second, depending on the temperature and pressure, the heating of the droplet may be counteracted by the loss of heat of vaporization of the volatile component. Therefore, care must be taken to ensure that the fluid droplets do not reach the end of the reactor and pool prior to being raised to the desired temperature, or that the temperature rise rate is insufficient. It may also be necessary to inject alcohol vapor with the steam to maintain equilibrium conditions in the reactor. It is noted that the reactor may also be used to reduce or vary alcohol concentrations of the fluid being treated, by varying the treatment conditions. For example, alcohol vapors may be withdrawn and captured through a vacuum pump, along with non-condensing gasses and some steam. This allows the production of a xe2x80x9clightxe2x80x9d alcoholic beverage, while killing yeast or other organisms.
It is also noted that various non-biological compositions may have glass transition temperatures in the 25-300xc2x0 C. temperature range, and therefore the present apparatus may be useful for using steam to rapidly alter a crystalline state of, e.g., these polymers, copolymers, block copolymers or interpenetrating polymer networks. This rapid limited heating may be advantageous, for example, to rapidly initiate a chemical reaction while maintaining a mechanical configuration of a bead, for example, if the time constant for the chemical reaction is comparatively fast with respect to the thermal diffusion time constant. Further, in larger droplets, an external polymerized shell may be formed around an unpolymerized interior.
Modification of Cells
The RTCP technique, under appropriate conditions, has the ability to alter cells, without significant denaturation of most proteins. There are three mechanisms postulated to exert effects on the cells:
(a) Heating
Cells are heated in a rapid (millisecond rise-time) and controlled manner to a desired temperature, and held for a short period. Cooling is slower, on the order of seconds. Millisecond heating causes two potential effects. The thermal expansion wave, as well as differential coefficients of thermal expansion, produce a mechanical stress directed on the membrane or cell wall. The cell membrane may also transition through a glass transition temperature faster than the cell can accommodate, resulting in membrane disruption. For example, the glass transition temperature of dipalmitoyl phosphatidyl choline, by scanning differential calorimetry, is about 41xc2x0 C. xe2x80x9cPhysical Properties and Functional Roles of Lipids in Membranesxe2x80x9d, Biochemistry of Lipids, Lipoproteins and Membranes (1996).
(b) Osmotic Stress
The heating effect is carried out by condensation of saturated water vapor on relatively cooler atomized droplets of medium. The dilution may be in the range of 1-10%, which occurs synchronously with the heating. This osmotic stress also acts on the membrane.
(c) Mechanical Shock
The medium, for treatment, is forced through an orifice, causing uniform atomization as a spray of droplets at high velocity, which pass through a steam chamber, reaching equilibrium temperature with the steam before hitting a distal wall. The atomization and deceleration after treatment both produce significant mechanical stresses on the cell.
In many medical treatments, it may be desired to avoid king the cells, but rather to exert a stress to which the cell responds. In this case, the RTCP system is tuned to provide a controlled thermal effect, limited osmotic stress (associated with the temperature rise), and minimized mechanical stress. Thus, mechanical cellular disruption, an effect which is available through other means, is minimized.
In contrast, where it is desirable to kill cells, each of the effects is maximized, limited by thermal denaturation effects and turbulent alterations of the media. In some cases, killing of a partial cell population may be sufficient, in which case the treatment conditions are modulated to minimize undesired side effects, such as dilution, turbulence, medium protein precipitation, etc. In other cases, it is desired to kill all of a cell population or sterilize the medium. In this case, the treatment conditions are established to kill the desired cells with a desired statistical margin.
It is therefore an object of the present invention to provide a system and method for fusing lipid membranes, comprising the steps of providing the membranes to be fused in a liquid medium; and heating the liquid medium, containing the membranes to be fused, at a rate and through a range sufficient to cause a discrete transition in at least one of the membranes, such that the membranes fuse.
It is also an object according to the present invention to provide a system and method for disrupting lipid bilayers, comprising the steps of providing the lipid bilayer in a polar medium; and heating the polar medium at a rate and through a range sufficient to generate a shock wave in the lipid bilayer to reduce an integrity thereof.
It is a further object according to the present invention to provide a system and method for fusing a liposome with a cell, comprising the steps of providing a liposome and a cell in mutual proximity in a physiological medium; and heating the physiological medium at a rate sufficient and through a range appropriate to cause an abrupt glass transition in a portion of at least one of the liposome and the cell to cause a fusion thereof.
It is also an object according to the present invention to provide a system and method for processing a lipid bilayer structure, comprising the steps of providing a lipid bilayer structure in a liquid polar medium; and heating the liquid polar medium at a rate sufficient and through a range appropriate to cause an abrupt glass transition in a portion of the lipid bilayer structure to alter a mechanical configuration thereof.
One of the membranes may be of a eukaryotic organism, for example a mammal. The cell is preferably a circulating formed blood component, such as an erythrocyte, lymphocyte or phagocyte, or even platelet. The cell may also be an abnormal cell, such as a malignant cell, immortalized cell, cell infected with a bacteria, virus or other intracellular parasite, a cell having a genetic or environmentally induced deficiency or surplus of mineral, nutrient, enzyme or other composition.
The system may be used to treat a single type of membrane-bound structure, a pair of structures, or a mixture of a number of structures. For example, cells may be fused with two or more types of liposomes.
The membrane may also be an engineered structure, such as a liposome or vesicle. The engineered structure may have a specific composition on its surface (outer or inner) or interior. The composition may be a pharmaceutical, nutrient, oxidant or antioxidant, cytokine, enzyme (e.g., glucose-6-dehydrogenase), protein, receptor, receptor binding ligand, receptor agonist or antagonist, hormone, gene regulatory agent, antibody or portion thereof, cytotoxic agent, redox state altering composition, pH altering composition, viral protein, viral receptor protein, nucleic acid (e.g., nucleic acid encoding at least one gene or regulator). For example, liposome containing phosphatidylethanolmines, diacylglycerol, ethanol, short chain fatty acids, and/or lipid peroxides be treated, for fusion with a cell.
After treatment, the product may be stored, for example for hours, days or longer, or further processed or employed in a medical treatment. The product may also be used in industrial or biotechnical processes. The cells an/or medium may be injected or infused into an animal for example intravenous or directed to lymphatic pathways.
During or in conjunction with heat treatment, the membranes or medium may be subject to other conditions, such as oxidizing or reducing agents, antioxidant (free radical trapping) agents, photonic or microwave radiation treatment, turbulence or shear forces, or the like. A non-thermal bactericidal treatment may therefore be applied in conjunction with the heat treatment. The medium may be filtered to remove most bacteria.
As a result of heat treatment, the membrane may be reversibly altered, irreversibly damaged, fused or other alterations, in addition to changes due to components added or removed from the membrane during the process. Thus, a cell may be killed or remain alive as a result of the treatment. One set of embodiments according to the invention achieves sterility, for example killing prokaryotic and eukaryotic cells, including mycoplasma.
According to one object of the invention, two different membrane structures are subjected to treatment, one membrane structure having an effective glass transition temperature below an average glass transition temperature of the other. One of the structures may be homogeneous while the other is heterogeneous, e.g., a mosaic domain structure as hypothesized by Singer and Nichols. The heat treatment my therefore induce a gel to liquid state transition in at least a portion of one membrane. The temperature rise rate may exceed an accommodation rate of the membrane.
The rapid heating may, for example, cause at least a portion of a lipid bilayer membrane to enter a non-bilayer state. The heating may also cause a non-linear change in packing density of molecules forming a membrane. In order to improve the efficiency or selectivity of the process, a cell may be incubated under such conditions as to alter a cell membrane lipid composition.
The temperature rise rate is, e.g., greater than 100 C per second, and may be greater than 1000 C per second. The temperature rise may be greater than 10 C, for example 25 to 40 C, and the maximum process temperature may be less than 55 C, preferably less than 49 C, and possibly as low or lower than 43 C.
The heat treatment preferably does not substantially denature cellular proteins, although under certain conditions, denaturation of at least certain proteins may be desired.
The medium may be a physiological solution, milk from a mammal human milk, milk or blood from a transgenic mammal blood plasma or serum, fermentation broth, water, saline or other fluids.
The heating may be effected, for example, by water vapor or steam, which is preferably xe2x80x9csuperheatedxe2x80x9d or xe2x80x9cdryxe2x80x9d to reduce spontaneous condensation. An inert, non-condensing gas may be present during treatment, or the treatment may be conducted with low non-condensing gas levels. Thus, non-condensing gasses may be added or removed during treatment. The medium is preferably degassed prior to treatment.
The medium is preferably atomized prior to heating. The atomized medium is preferably heated while moving at a velocity of at least about 10 cm/sec, preferably at least 100 cm/sec, and more preferably 1000 to 2000 cm/sec or higher. The medium may be subject to mechanical forces synchronized with and independent of the heating.
The apparatus has, for example, a processing capacity of between about 0.25-125 ml per minute, and preferably a processing capacity of between about 1-25 ml per minute. The apparatus may have a transparent treatment chamber, for example made of glass, e.g., borosilicate glass or of fused quartz. The apparatus may also include an automated or assisted cleaning cycle to achieve sterilization and/or to remove deposits. The apparatus preferably has a control, the control being programmed to detect a treatment aberration.
In one embodiment of the invention, two cells are fused in order to achieve a hybrid. Such a process includes treatment of a malignant or immortalized cell and differentiated cell to result in a cell having differentiated characteristics, such as specific gene products, e.g., a significant secreted gene product, such as an antibody. The resulting cell line may therefore produce, e.g., a monoclonal antibody from an immunoglobulin secreting hybridoma.
Other objects and advantages of the present invention will become apparent from a review of the drawings and detailed description.